The present invention relates to the field of cellular biology and more particularly, to an improved device and method for the study of cells. Specifically, the present invention is a method and a device for identification of the image of individual wells in an image of a well-bearing component so as to allow efficient image analysis and signal detection of cells held in the wells.
Combinatorial methods in chemistry, cellular biology and biochemistry are essential for the near simultaneous preparation of multitudes of active entities such as molecules. Once such a multitude of molecules is prepared, it is necessary to study the effect of each one of the active entities on a living organism.
The study of the effects of stimuli such as exposure to active entities on living organisms is preferably initially performed on living cells. Since cell-functions include many interrelated pathways, cycles and chemical reactions, the study of an aggregate of cells, whether a homogenous or a heterogeneous aggregate, does not provide sufficiently detailed or interpretable results: rather a comprehensive study of the biological activity of an active entity may be advantageously performed by examining the effect of the active entity on a single isolated living cells. Thus, the use of single-cell assays is one of the most important tools for understanding biological systems and the influence thereupon of various stimuli such as exposure to active entities.
The combinatorial preparation of a multitudes of active entities coupled with the necessity of studying the effect of each one of the active entities on living organisms using a single-cell assay, requires the development of high-throughput single live cell assays. There is a need for the study of real-time responses to treatment in large and heterogeneous cell populations at an individual cell level. In such studies it is essential to have the ability to define multiple characteristics of each individual cell, as well as the individual cell response to the experimental intervention of interest.
In the art, various different methods for studying living cells are known.
Multiwell plates having 6, 12, 48, 96, 384 or even 1536 wells on a standard ca. 8.5 cm by ca. 12.5 cm footprint are well known in the art. Such multiwell plates are provided with an 2n by 3n array of rectangular packed wells, n being an integer. The diameter of the wells of a plate depends on the number of wells and is generally greater than about 250 microns (for a 1536 well plate). The volume of the wells depends on the number of wells and the depth thereof but generally is greater than 5×10−6 liter (for a 1536 well plate). The standardization of the formats of multiwell plates is a great advantage for researchers as the standardization allows the production of standardized products including robotic handling devices, automated sample handlers, sample dispensers, plate readers, observation components, plate washers, software and such accessories as multifilters.
Multiwell plates are commercially available from many different suppliers. Multiwell plates made from many different materials are available, including but not limited to glass, plastics, quartz and silicon. Multiwell plates having wells where the inside surface is coated with various materials, such as active entities, are known.
Although exceptionally useful for the study of large groups of cells, multiwell plates are not suitable for the study of individual cells or even small groups of cells due to the large, relative to the cellular scale, size of the wells. Cells held in such wells either float about a solution or adhere to a well surface. When cells float about in a well, specific individual cells are not easily found for observation. When cells adhere to a well surface, the cells adhere to any location in the well, including anywhere on the bottom of the well and on the walls of the well. Such variability in location makes high-throughput imaging (for example for morphological studies) challenging as acquiring an individual cell and focusing thereon is extremely difficult. Such variability in location also makes high-throughput signal processing (for example, detection of light emitted by a single cell through fluorescent processes) challenging as light must be gathered from the entire area of the well, decreasing the signal to noise ratio. Further, a cell held in a well of a multiwell plate well can be physically or chemically manipulated (for example, isolation or movement of a single selected cell or single type of cell, changing media or introducing active entities) only with difficulty. Further, the loading of multiwell plates as expressed in terms of cells held singly in the wells per unit area is very low (about 1536 cells in 65 cm2, or 24 cells cm−2) Thus, multiwell plates are in general only suitable for the study of homogenous or heterogenous aggregates of cells as a group.
An additional disadvantage of multiwell plates is during the study of cells undergoing apoptosis. One method of studying cells is by exposing cells in a monolayer of cells adhered to the bottom of the well of a multiwell plate to a stimulus. As is known to one skilled in the art, one of the most important processes that a cell potentially undergoes is apoptosis and it is highly desirable to observe a cell throughout the apoptosis process. However, once a cell begins the apoptosis process, the adhesion of the cell to the bottom of the well is no longer sufficient: the cell detaches from the bottom and is carried away by incidental fluid currents in the well. The cell is no longer observable and its identity lost.
An additional disadvantage of multiwell plates is in the study of non-adhering cells. Just as cells undergoing apoptosis, in multiwell plates non-adhering cells can be studied as individuals only with difficulty. Considering that non-adhering cells are crucial for research in drug discovery, stem cell therapy, cancer and immunological diseases detection, diagnosis, therapy this is a major disadvantage. For example, blood contains seven heterogeneous types of non-adherent cells, all which perform essential functions, from carrying oxygen to providing immunity against disease.
In the art, a number of method and devices have been developed for the study of individual cells or a small number of cells as a group. Many such methods are based on using picowell-bearing device. A picowell-bearing device is a device for the study of cells that has at least one picowell-bearing component for study of cells. A picowell-bearing component is a component having at least one, but generally a plurality of picowells, each picowell configured to hold at least one cell. The term “picowell” is general and includes such features as dimples, depressions, tubes and enclosures. Since cells range in size from about 1 microns to about 100 (or even more) microns diameter there is no single picowell size that is appropriate for holding a single cell of any type. That said, the dimensions of the typical individual picowell in the picowell-bearing components known in the art have dimensions of between about 1 microns up to about 200 microns, depending on the exact implementation. For example, a device designed for the study of single isolated 20 micron cells typically has picowells of dimensions of about 20 microns. In other cases, larger picowells are used to study the interactions of a few cells held together in one picowell. For example, a 200 micron picowell is recognized as being useful for the study of the interactions of two or three cells, see PCT patent application IL01/00992 published as WO 03/035824.
One feature that increases the utility of a picowell-bearing device is that each individual picowell is individually addressable. By individual addressability is meant that each picowell can be registered, found or studied without continuous observation. For example, while cells are held in picowells of a picowell-bearing component, each cell is characterized and the respective picowell where that cell is held is noted. When desired, the observation component of the picowell-bearing device is directed to the location of the picowell where a specific cell is held. One method of implementing individual addressability is by the use of fiducial points or other features (such as signs or labels), generally on the picowell-bearing component. Another method of implementing individual addressability is by arranging the picowells in a picowell-array and finding a specific desired picowell by counting. Another method of implementing individual addressability is by providing a dedicated observation component for each picowell.
In the art, the picowell-bearing component of a picowell-bearing device is often a chip, a plate or other substantially planar component. Herein such a component is termed a “carrier”. In the art, there also exist non-carrier picowell-bearing components of picowell-bearing devices, for example, bundles of fibers or bundles of tubes.
Mrksich and Whitesides, Ann. Rev. Biophys. Biomol. Struct. 1996, 25, 55-78; Craighead et al., J. Vac. Sci. Technol. 1982, 20, 316; Singhvi et al., Science 1994, 264, 696-698; Aplin and Hughes, Analyt. Biochem. 1981, 113, 144-148 and U.S. Pat. No. 5,324,591 all teach of devices including arrays of spots of cell-attracting or cell-binding entities on a plate. In such devices, the spots serve as picowells, binding cells through a variety of chemical bonds. In such devices, the plate is the picowell-bearing component of the device. Due to the size of the spots, each such picowell generally holds more than one cell. To reduce interaction between cells held at different picowells, the spots must be spaced relatively far apart, reducing loading as expressed in terms of picowells per unit area. Even with generous spacing, in such picowell-bearing components held cells are not entirely isolated from mutual interaction, nor can cells be subject to individual manipulation. The fact that the cells are not free-floating but are bound to the plate through some interaction necessarily compromises the results of experiments performed.
In U.S. Pat. No. 6,103,479, the picowell-bearing component is a transparent carrier provided with a non-uniform array of picowells, each well functionalized with chemical entities that bind to cells specifically or non-specifically. Each picowell is of approximately 200 to 1000 micron diameter and is configured to hold a plurality of cells. The inter picowell areas are hydrophobic so as not to attract cells. In addition to the carrier, a device of U.S. Pat. No. 6,103,479 is provided with a glass, plastic or silicon chamber-bearing plate in which individually addressable microfluidic channels are etched, the chamber-bearing plate configured to mate with the carrier. When mated, the carrier and chamber-bearing plate constitute a cassette in which each cell is bound to the carrier and isolated in a chamber provided with an individual fluid delivery system. Reagents are provided through the fluid delivery system and observed by the detection of fluorescence. In order to provide space for the walls of the chambers, the inter picowell areas of the carrier are relatively large, reducing loading as expressed in terms of picowells per unit area. Subsequent to study, the cassette is separated into the two parts and the micro-patterned array of cells processed further. In some embodiments, the chamber-bearing plate is made of polytetrafluoroethylene, polydimethylsiloxane or an elastomer. As held cells do not make contact with the chamber-bearing plate it is not clear what advantages are to be had when providing a chamber-bearing plate of such esoteric materials.
In U.S. patent application Ser. No. 10/199,341, a device is taught for trapping a plurality of dielectric objects (such as cells), each individual object in an individual light beam produced by an optical array.
In U.S. Pat. No. 4,729,949, a device is taught for trapping individual cells in a picowell-bearing carrier, the carrier being substantially a plate having a plurality of picowells that are individually-addressable tapered apertures of a size to hold individual cells. Suction applied from the bottom surface of the plate where the picowells are narrow creates a force that draws cells suspended in a fluid above the carrier into the wide end of the picowells on the surface of the carrier to be held therein. Using the teachings of U.S. Pat. No. 4,729,949 a specific group of cells (having dimensions similar to that of the wide end of the picowells) can be selected from amongst a group of cells and held in the carrier. Although the cells are subjected to common stimuli, the fact that the picowells are individually addressable allows the effect of a stimulus on an individual cell to be observed. A carrier of U.S. Pat. No. 4,729,949, is generally made of metal such as nickel and prepared using standard photoresist and electroplating techniques. In a carrier of U.S. Pat. No. 4,729,949, the inter picowell areas of the carrier are relatively large, leading to a low loading as expressed in terms of picowells per unit area. Further, the suction required to hold cells in picowells of a carrier of U.S. Pat. No. 4,729,949 deforms held cells and makes a significant portion of the cell membranes unavailable for contact, both factors that potentially compromise experimental results. Study of cells with non-fluorescence based methods generally gives poor results due to reflections of light from the carrier.
In PCT patent application U.S.99/04473 published as WO 99/45357 is taught a picowell-bearing component produced by etching the ends of a bundle of optical fibers (apparently of glass) while leaving the cladding intact to form a picowell-bearing component that is a bundle of tubes. The size of the hexagonal picowells is demonstrated to be as small as 7 micron wide, 5 micron deep and having a volume of about 1.45×10−13 liter. The inter picowell area is quite large due to the thickness of the cladding of the optical fibers. Light emitted by cells held in each picowell are independently observable through a respective optical fiber. In some embodiments, the inside surface of the picowells is coated with a film of materials such as collagen, fibronectin, polylysine, polyethylene glycol, polystyrene, fluorophores, chromophores, dyes or a metal. Loading the picowell-bearing component of PCT patent application U.S.99/04473 includes dipping the optical fiber bundle in a cell suspension so that cells adhere to the picowells. There are a number of disadvantages to the teachings of PCT patent application U.S.99/04473. The fact that the cells are studied only subsequent to adhesion to the picowells necessarily influences the results of experiments performed. Since cell proliferation generally begins soon after adhesion, it is not known if a detected signal is produced by a single cell or a plurality of cells. It is is not clear where exactly in a picowell a cell is held and therefore what percentage of light emitted from a cell travels to a detector. The fact that emitted light travels through an optical fiber leads to loss of time-dependent and phase information.
In unpublished copending PCT patent application IL04/00192 of the Applicant filed Feb. 26, 2004 is taught a picowell-bearing component produced by bundling together glass capillaries, each glass capillary attached to an independent fluid flow generator such as a pump. A cell held in a first picowell is transferred to a second picowell by the simultaneous application of an outwards flow from the first picowell and an inwards flow into the second picowell.
A preferred device for the study of cells is described in PCT patent application IL01/00992 published as WO 03/035824 of the Applicant. The device 10, depicted in FIG. 1, is provided with a transparent carrier 12 as a picowell-bearing component. Carrier 12 is substantially a sheet of transparent material (such as glass or polystyrene) on the surface of which features such as inlet connectors 14, fluid channels 16, picowells (in FIG. 1, a picowell-array 18), a fluid reservoir 20 and an outlet connector 22. Carrier 12 is immovably held in a holder 24 having a cutout window of a size and shape to accept carrier 12. Other components of device 10 not depicted include flow generators, observation components, external tubing and the like. When a cover slip (not depicted) is placed or integrally formed with carrier 12, fluid channels 16, picowell-array 18 and reservoir 20 are sealed forming channels that allow transport of fluids and reagents to cells held in picowell-array 18. The picowells are configured to hold a predetermined number of cells (one or more) of a certain size and are preferably individually addressable both for examination and manipulation.
FIG. 2 is a reproduction of a photograph of a different carrier 26 held in a holder 24. A first syringe 28 as an inlet flow generator is in communication with an inlet connector 14 by a capillary tube 30. Inlet connector 14 is in communication with picowell-array 18 through a fluid passage 16. Picowell-array 18 is in communication with outlet connector 22 through a fluid passage 16. A second syringe 32 as an outlet flow generator is in communication with outlet connector 22 through capillary tube 34.
PCT patent application IL01/00992 also teaches methods of physically manipulating cells held in a picowell-bearing device using, for example, individually addressable microelectrodes (found in the picowells or in the cover slip) or optical tweezers. Typical physical manipulations include moving selected cells into or out of specific picowells. One useful method that is implemented using a device of PCT patent application IL01/00992 is that cells, each held alone in a respective picowell, are examined (either in the presence or absence of reagents) and based on the results of the examination, cells with a certain characteristic are selected to remain in a respective picowell while cells without the certain characteristic are removed from a respective picowell and ejected by the application of a flow in parallel to the surface of the carrier, generated by a flow generator.
An additional feature of the teachings of PCT patent application IL01/00992 is that, in some embodiments, the picowells are juxtaposed, that is, the area occupied by a picowell-array is substantially entirely made up of picowells with little or no inter picowell area, see FIG. 3. FIG. 3 is a reproduction of a photograph of part of a picowell-array 18 from the top of a carrier 12 of PCT patent application IL01/00992. In FIG. 3 is seen a plurality of hexagonal picowells 36, some populated with living cells 38. It is seen that the inter picowell areas 40 make up only a minor percentage of the total area of picowell-array 18. This feature allows near tissue-density packing of cells, especially in single-cell picowell configurations. For example, a typical device of PCT patent application IL01/00992 having a 2 mm by 2 mm picowell-array of hexagonally-packed juxtaposed picowells of 10 micron diameter and no inter picowell area includes about 61600 picowells. This feature also allows simple picowell loading: a fluid containing suspended cells is introduced in the volume above the picowells. Since there is little inter picowell area, cells settle in the picowells.
One of the challenges of well-bearing devices known in the art for the study of single living cells, especially picowell-bearing devices, is of information acquisition.
One type of information acquisition is manual image analysis. Manual image analysis involves a cell biology expert visually inspecting cells, for example using an observation component equipped with optical magnification means such as a microscope and drawing conclusions based on the visual inspection. Manual image analysis is time-consuming, incompatible with high-throughput studies and is not generally applicable.
Two other type of information acquisition are automatic image analysis and automatic signal acquisition.
In automatic image analysis, high-resolution optical data is acquired substantially continuously for all wells of interest and cells held therein. A disadvantage of using automatic image analysis is that there is no easy way to sift through the massive amount of information acquired to identify important events from amongst all the images acquired.
In automatic signal analysis, one or limited number of signal channels, usually corresponding to a light intensity, are acquired as a function of time for each well and cells held therein substantially continuously. Often, the signal channels acquired correspond to different wavelengths of light emitted by fluoresence processes occuring in the wells.
One of the greatest challenges in both automatic image analysis and automatic signal analysis is the delineation of the borders of a single cell. For example, in FIG. 4 is depicted a reproduction of a transparent light image of MALT-4 cells on a glass plate. Individual cells and borders thereof were automatically determined. In many cases, cells are not identified. For example, in the upper left corner of FIG. 4, an aggregate of three cells designated “159” is identified to be one cell. In the middle right side of FIG. 4, the borders of cells designated as “439” and “438” are improperly delineated. In both such cases, analysis of an image or of a signal gives completely wrong results. Even when cells are held in picowells 36, for example, as depicted in FIG. 3, it is difficult to delineate the borders of wells and of cells held therein, especially when slight shifting of the picowell-bearing component relative to the field of view occurs, whether due to physical motion of the picowell-bearing component or as a result of motion of the observation component. Further, due to the fact that the material from which wells are made is not invisible, distortions, reflections, diffractions and the image of the picowell walls often make delineation of cells difficult. For example, differentiating cell 42 from cell 44 in FIG. 3 is a difficult task. It is important to note that even the imperfect methods known in the art are time consuming, expensive in terms of calculation resources, not robust and in general unsuited for high-throughput applications.
The problem of delineating the borders of a cell for automatic signal analysis is even greater. When automatic signal analysis is implemented, it is desired that the implementation be quick, robust and is directed for high-throughput analysis of many cells. In such applications, it is not practical to have a time consuming cell-identification or picowell-identification step. In addition, if the borders of the cell or picowells are not clearly delineated, the quality of the data is seriously compromised. For example, when a cell is delineated conservatively, and only a portion of a signal emitted by a cell is acquired the values of the acquired signal will be innacurate, especially in cases where signals are not emitted from all areas of a cell homogenously. For example, when a cell is delineated too broadly and signals from neighboring cells are also acquired the signal to noise ratio decreases. An additional problem arises when what is to be detected is not light emitted by a cell itself but rather light emitted by chromatogenic or fluorogenic entitities in the medium in the immediate area of the cell, for example the medium held together with the cell in the same picowell. In such experiments it is critical to know the exact borders of the picowell in which a cells is held.
In the art, a number of solutions based on providing each well with a dedicated observation system have been proposed.
As discussed above in PCT patent application U.S.99/04473 is taught a picowell-bearing component produced by etching the ends of a bundle of optical fibers to form a picowell-bearing component where a cell held inside such a picowell necessarily is associated with an adressable optical fiber that tranports light emitted from the picowell to a detector for signal acquisition. As stated above, amongst other problems associated with the device of PCT patent application U.S.99/04473, the fact that the emitted light travels through an optical fiber leads to loss of time dependent and phase information. Further, the device of PCT patent application U.S.99/04473 is not suitable for acquiring high-resolution images.
A preferred method of automatic image acquisition where a well and the contents thereof are clearly delineated is described, for example, in PCT patent application IL01/000992 where in one embodiment is taught a device having an individual microlens dedicated to the continuous observation of every picowell of the picowell-bearing component and cells held therein. Such a method requires a highly expensive observation system, including a dedicated, accurately crafted and expensive microlens array. Further, such a microlens array must be located above the picowell array and is generally exposed to the medium in which cells are held.
It would be highly advantageous to have a device and methods for the study of cells not having at least some of the disadvantages of the prior art.